PCR differentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using specific primers

The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5'-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3'-SSU end) and the 3'-specific primers were located in the LSU gene (positions 560-590 from the 5'-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.     

Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR

AIMS: To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. METHODS AND RESULTS: Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. CONCLUSIONS: Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.     

Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolated from Amarone wine

Individual yeast strains belonging to the Saccharomyces sensu stricto complex were isolated from Amarone wine produced in four cellars of the Valpolicella area (Italy) and characterized by conventional physiological tests and by RAPD-PCR and mtDNA restriction assays. Thirteen out of 20 strains were classified as Saccharomyces cerevisiae (ex S. cerevisiae p.r. cerevisiae and p.r. bayanus) and the remaining as Saccharomyces bayanus (ex S. cerevisiae p.r. uvarum). RAPD-PCR method proved to be a fast and reliable tool for identification of Saccharomyces sensu stricto strains and also gave intraspecific differentiation. Restriction analysis of mtDNA permitted to distinguish S. cerevisiae and S. bayanus species and to discern polymorphism among S. cerevisiae isolates. The assessment of the phenotypic diversity within the isolates by gas-chromatographic analysis of secondary fermentation products was explored. Small quantities of isobutanol were produced by most of the strains and higher amounts by some S. cerevisiae strains with phenotypes Gal^- and Mel^-; all S. bayanus strains produced low amounts of amilyc alcohols. From this study it appears that each winery owns particular strains, with different genetic and biochemical characteristics, selected by specific environmental pressures during the Amarone winemaking process carried out at low temperature in presence of high sugar content.     

The inheritance of mtDNA in lager brewing strains

In this work, we compared the mtDNA of a number of interspecific Saccharomyces hybrids (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces bayanus) to the mtDNA of 22 lager brewing strains that are thought to be the result of a natural hybridization between S. cerevisiae and another Saccharomyces yeast, possibly belonging to the species S. bayanus. We detected that in hybrids constructed in vitro, the mtDNA could be inherited from either parental strain. Conversely, in the lager strains tested, the mtDNA was never of the S. cerevisiae type. Moreover, the nucleotide sequence of lager brewing strains COXII gene was identical to S. bayanus strain NBRC 1948 COXII gene. MtDNA restriction analysis carried out with three enzymes confirmed this finding. However, restriction analysis with a fourth enzyme (AvaI) provided restriction patterns for lager strains that differed from those of S. bayanus strain NBRC 1948. Our results raise the hypothesis that the human-driven selection carried out on existing lager yeasts has favored only those bearing optimal fermentation characteristics at low temperatures, which harbor the mtDNA of S. bayanus.     

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

Mitochondrial DNA polymorphism of the yeast Saccharomyces bayanus var. uvarum

Genetic relationships among forty-one strains of Saccharomyces bayanus var. uvarum isolated in different wine regions of Europe and four wild isolates were investigated by restriction analysis (RFLP) of mitochondrial DNA (mtDNA) with four restriction endonucleases, AluI, DdeI, HinfI and RsaI. No clear correlation between origin and source of isolation of S. bayanus var. uvarum strains and their mtDNA restriction profiles was found. On the whole, the mtDNA of S. bayanus var. uvarum is much less polymorphic than that of S. cerevisiae. This observation is in good agreement with results obtained by electrophoretic karyotyping. Unlike wine S cerevisiae, strains of S. bayanus var. uvarum display a low level of chromosome length polymorphism.     

Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

Nine yeast strains were isolated from spontaneous fermentations in the Alsace area of France, during the 1997, 1998 and 1999 grape harvests. Strains were characterized by pulsed-field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) of the MET2 gene, delta-PCR, and microsatellite patterns. Karyotypes and MET2 fragments of the nine strains corresponded to mixed chromosomal bands and restriction patterns for both Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum. They also responded positively to amplification with microsatellite primers specific to both species and were demonstrated to be diploid. However, meiosis led to absolute nonviability of their spores on complete medium. All the results demonstrated that the nine yeast strains isolated were S. cerevisiaexS. bayanus var. uvarum diploid hybrids. Moreover, microsatellite DNA analysis identified strains isolated in the same cellar as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.     

A multispecies-based taxonomic microarray reveals interspecies hybridization and introgression in Saccharomyces cerevisiae

A multispecies-based taxonomic microarray targeting coding sequences of diverged orthologous genes in Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Saccharomyces kudriavzevii, Naumovia castellii, Lachancea kluyveri and Candida glabrata was designed to allow identification of isolates of these species and their interspecies hybrids. Analysis of isolates of several Saccharomyces species and interspecies hybrids demonstrated the ability of the microarray to differentiate these yeasts on the basis of their specific hybridization patterns. Subsequent analysis of 183 supposed S. cerevisiae isolates of various ecological and geographical backgrounds revealed one misclassified S. bayanus or Saccharomyces uvarum isolate and four aneuploid interspecies hybrids, one between S. cerevisiae and S. bayanus and three between S. cerevisiae and S. kudriavzevii . Furthermore, this microarray design allowed the detection of multiple introgressed S. paradoxus DNA fragments in the genomes of three different S. cerevisiae isolates. These results show the power of multispecies-based microarrays as taxonomic tools for the identification of species and interspecies hybrids, and their ability to provide a more detailed characterization of interspecies hybrids and recombinants.     

Molecular typing of wine yeast strains Saccharomyces bayanus var. uvarum using microsatellite markers

The Saccharomyces bayanus var. uvarum yeasts are associated with spontaneous fermentation of must. Some strains were shown to be enological yeasts of interest in different winemaking processes. The molecular typing of S. bayanus var. uvarum at the strain level has become significant for wine microbiologists. Four microsatellite loci were defined from the exploration of genomic DNA sequence of S. bayanus var. uvarum. The 40 strains studied were homozygote for the locus considered. The discriminating capacity of the microsatellite method was found to be equal to that of karyotypes analysis. Links between 37 indigenous strains with the same geographic origin could be established through the analysis of microsatellite patterns. The analysis of microsatellite polymorphism is a reliable method for wine S. bayanus var. uvarum strains and their hybrids with Saccharomyces cerevisiae identification in taxonomic, ecological studies and winemaking applications.     

A novel specific DNA marker in Saccharomyces bayanus for species identification of the Saccharomyces sensu stricto complex

This study was based on RAPD fingerprinting for species identification of the Saccharomyces sensu stricto complex. 40 random primers were used for RAPD analysis. The results showed that one of these primers, OPT-18, produced a 974 bp species-specific band, which was only found in the tested S. bayanus. Afterward this specific fragment was isolated from agarose gel and ligated into vector for DNA sequencing. A pair of primer SpeOPT18Sbay-F2 and SpeOPT18Sbay-R2 were designed according to the cloned species-specific sequence, which was employed for PCR with the template DNA of the S. sensu stricto strains, single 779 bp species-specific band was only found in S. bayanus. Therefore, we conclude that our novel species DNA marker could be used to rapidly and accurately identify the species of S. bayanus from S. sensu stricto complex by direct PCR.     

Temperature adaptation markedly determines evolution within the genus Saccharomyces

The present study uses a mathematical-empirical approach to estimate the cardinal growth temperature parameters (T(min), the temperature below which growth is no longer observed; T(opt), the temperature at which the μ(max) equals its optimal value; μ(opt), the optimal value of μ(max); and T(max), the temperature above which no growth occurs) of 27 yeast strains belonging to different Saccharomyces and non-Saccharomyces species. S. cerevisiae was the yeast best adapted to grow at high temperatures within the Saccharomyces genus, with the highest optimum (32.3°C) and maximum (45.4°C) growth temperatures. On the other hand, S. kudriavzevii and S. bayanus var. uvarum showed the lowest optimum (23.6 and 26.2°C) and maximum (36.8 and 38.4°C) growth temperatures, respectively, confirming that both species are more psychrophilic than S. cerevisiae. The remaining Saccharomyces species (S. paradoxus, S. mikatae, S. arboricolus, and S. cariocanus) showed intermediate responses. With respect to the minimum temperature which supported growth, this parameter ranged from 1.3 (S. cariocanus) to 4.3°C (S. kudriavzevii). We also tested whether these physiological traits were correlated with the phylogeny, which was accomplished by means of a statistical orthogram method. The analysis suggested that the most important shift in the adaptation to grow at higher temperatures occurred in the Saccharomyces genus after the divergence of the S. arboricolus, S. mikatae, S. cariocanus, S. paradoxus, and S. cerevisiae lineages from the S. kudriavzevii and S. bayanus var. uvarum lineages. Finally, our mathematical models suggest that temperature may also play an important role in the imposition of S. cerevisiae versus non-Saccharomyces species during wine fermentation.     

The molecular genetic differentiation of cultured Saccharomyces strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that baker's yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae and S. bayanus var. uvarum hybrids. The molecular karyotyping of baker's, brewer's, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)5 can be used to study the populations of cultured S. cerevisiae strains.     

Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains

AIMS: To assess, for the first time the efficiency in removing ochratoxin A (OTA) from laboratory medium [yeast peptone glucose (YPG)], synthetic grape juice medium (SGM) and natural grape juice by viable and dead (heat and acid-treated) oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) compared with a commercial yeast walls additive. METHODS AND RESULTS: Levels of OTA during its interaction with six oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) or with a commercial yeast walls additive in YPG medium, in SGM or in natural grape juices was assessed by HPLC after appropriate extraction methods. A significant decrease of OTA levels in YPG medium and SGM was observed for many of the growing strains reaching a maximum of 45%, but no degradation products were detected. With both heat and acid pretreated yeasts, OTA removal was enhanced, indicating that adsorption, not catabolism, is the mechanism to reduce OTA concentrations. Adsorption was also improved when the yeast concentration was increased and when the pH of the medium was lower. Approximately 90% of OTA was bound rapidly within 5 min and up to 72 h of incubation with heat-treated cells of either S. cerevisiae or S. bayanus. A comparative study between heat-treated cells (HC) and commercial yeast walls (YW) (used as oenological additive), introduced at two different concentrations (0.2 and 6.7 g l(-1)) in an OTA-contaminated grape juice, showed the highest efficiency by HC to adsorb rapidly within 5 min the total amount of the mycotoxin. CONCLUSIONS: Oenological S. cerevisiae and S. bayanus were able to remove ochatoxin A from synthetic and natural grape juices. This removal was rapid and improved by dead yeasts having more efficiency than commercial yeast walls. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficiency of heat-treated yeasts to remove OTA gives a new hope for grape juice and must decontamination avoiding negative impacts on human health.     

Characterisation of thermotolerant Saccharomyces cerevisiae hybrids

Thermotolerant Saccharomyces strains were crossed with mesophilic Saccharomyces cerevisiae and with cryotolerant Saccharomyces bayanus. The former hybrid is fertile confirming thermotolerant strains as S. cerevisiae. The spores from this hybrid, though, possess a low germinability and give cultures that grow poorly. The hybrid cryotolerant x thermotolerant is sterile and show a new combination of the parental strains' traits improving their technological application. © Rapid Science Ltd. 1998     

Evolutionary relationships between the former species Saccharomyces uvarum and the hybrids Saccharomyces bayanus and Saccharomyces pastorianus; reinstatement of Saccharomyces uvarum (Beijerinck) as a distinct species

Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.     

Ribosomal DNA restriction analysis reveals genetic heterogeneity in Saccharomyces cerevisiae Meyen ex Hansen.

A ribosomal DNA restriction analysis of 17 strains belonging to the Saccharomyces cerevisiae complex revealed two major groups, one of which corresponded to Saccharomyces bayanus. Our results generally correlate with previously reported genetic and molecular data and support the conclusion that S. bayanus should be reinstated as a separate taxon.     

A molecular genetic study of natural strains of Saccharomyces isolated from Asturian cider fermentations

AIMS: To analyse the genetic diversity and the dynamics of Saccharomyces strains in spontaneous fermentation in ciders. The effect of the cellar, harvest and cider-making technology were evaluated. METHODS AND RESULTS: The ecology of spontaneous cider fermentations in the same cellar (Asturias) was studied for two consecutive harvests (2000 and 2001) by using mtDNA restriction analysis. Our results showed that there was a succession of genetically different strains of Saccharomyces during cider production. In general, strains of Saccharomyces bayanus species predominated at the early fermentation steps (begining and/or tumultuous fermentations), while Saccharomyces cerevisiae yeasts were the most abundant at the end of the fermentation. Five S. bayanus strains (patterns III, VII, VIII, XV and XVII) were present at significant frequencies in all the experimental tanks during the two consecutive years. The results of the cluster analysis (unweighted pair group method using average linkage) showed higher similarities for the patterns III, XV, VII and VIII. Therefore, these strains should be considered associated with the microbiota of this cellar. CONCLUSIONS: A high polymorphism within populations of Saccharomyces was found throughout the different stages of Asturian production of cider. In all the cider fermentations, a variable number of S. bayanus and S. cerevisiae strains was always present. Our results indicate, over the period of time studied, the existence of the natural microbiota in the cellar. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of wild Saccharomyces yeast in Asturian cider fermentations.     

A structural and phylogenetic study of the HO gene from Saccharomyces bayanus var. uvarum

A novel HO gene (Uv-HO) was cloned from the Saccharomyces bayanus var. uvarum (abbreviated as S. uvarum in this study) type strain. The coding region of Uv-HO showed relatively high homology (95%) to that of the Sb-HO gene (S. bayanus var. bayanus HO), but not to the HO genes of other Saccharomyces sensu stricto species. However, the 5' and 3' non-coding region of Uv-HO showed less similarity (79% and 76% respectively) even to those of the most homologous gene Sb-HO. Motifs of the mating-type control and the cell-cycle control were conserved in the 5' non-coding region of Uv-HO, but numbers and positions of motifs were different from those of Sb-HO. CHEF-Southern analysis showed that all tested strains of S. bayanus species, including S. uvarum, carried the HO gene on the 1,100-kb chromosome. By HO-typing PCR using mixed primers, which provided a rapid and convenient tool for yeast identification, either the Uv-HO gene or the Sb-HO gene was detected in strains of S. bayanus species, but two strains were found to have both types of HO gene in each genome. These results suggest that S. uvarum has a unique sequence, but might share the same chromosome constitution within S. bayanus species, and that S. bayanus is a heterogeneous species, of which some strains might be natural hybrid.     

Isolation and characterization of the HO gene from the yeast Saccharomyces paradoxus

A DNA fragment homologous to the homothallism (HO) gene of Saccharomyces cerevisiae was isolated from Saccharomyces paradoxus and was found to contain an open reading frame that was 90.9% identical to the coding sequence of the S. cerevisiae HO gene. The putative HO gene was shown to induce diploidization in a heterothallic haploid strain from S. cerevisiae. Phylogenetic analysis revealed that the coding and 5'-upstream regulatory regions from five Saccharomyces sensu stricto HO genes have coevolved, and that S. paradoxus is phylogenetically closer to S. cerevisiae than to S. bayanus. Finally, heterothallic haploid strains were isolated from the original homothallic type strain of S. paradoxus by disrupting the S. paradoxus HO gene with the S. cerevisiae URA3 gene.     

Divergence of glyceraldehyde-3-phosphate dehydrogenase isozymes in Saccharomyces cerevisiae complex

Although sake yeasts are placed in Saccharomyces cerevisiae, we have been interested in their difference from the other subgroups of the species, and examined their proteins. When SDS-PAGE patterns of their soluble proteins were compared, specific differences between subgroups were found in their 36,000 Da regions. Proteins isolated therefrom were found to be subunits of three isomers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from their N-terminal amino acid sequences and identified with anti-GAPDH serum. Therefore, comparison of zymogram was carried out by a modified method: denatured monomers were observed and the enzyme activity of their oligomers was not considered. SDS-PAGE patterns of all the sake yeasts differed from those of the other strains of S. cerevisiae. Strains of Saccharomyces bayanus showed uniform patterns which are different from the above two groups. Saccharomyces pastorianus strains resembled S. bayanus and were partly similar to S. cerevisiae in their patterns, in agreement with the hypothesis that S. pastorianus is a hybrid between these two species. Patterns of S. paradoxus appeared to be rather similar to those of sake yeasts. Results on the other species of the genus and on the preliminary experiments on PAGE of native isozymes are also described.